Preparation of the Pituitary Gorticotrophic Hormone. ∗

Abstract

The procedure used for the preparation of the corticotrophic hormone from whole and from dissected calf anterior pituitary tissue was based on methods recommended by Lyons, by Li, Simpson and Evans, and by Sayers, White and Long. 1 , 2 , 3 It has been our experience that most of the posterior lobe potency present in the starting material remains with the fraction showing corticotrophic'activity. Attempts made to separate these factors by their solubilities at various pH and salt concentrations were without success. The same held true in utilizing dialysis with membranes of various porosities. This led us to attempt inactivation of the posterior lobe hormones with alkali. 4 Treatment with NaOH under varying conditions of concentration and time, when successful in removing posterior lobe activities, almost invariably resulted in appreciable losses of cortico-trophic potency. The same held true for NH₄OH treatment at higher temperatures. NH₄OH treatment at 37° and at 25°C resulted in the inactivation of most of the posterior lobe hormones without any appreciable loss of corticotrophic activity (Table I).

The method utilized for the preparation of the corticotrophic hormone containing only traces of posterior lobe contamination was as follows: (1) An 80% acidified acetone extraction was made from thoroughly minced tissue. Two such extractions, 6 hours each, were sufficient to remove all the active material; the residues being subjected to pressures in each instance. This was carried out at room temperatures, all subsequent procedures were done at 0° to 3° C. (2) The active material was precipitated from the combined filtrate by raising the acetone concentration to 90%. This was thoroughly washed with cold acetone and dried. (3) The powder, containing the corticotrophic and prolactin hormones, was extracted repeatedly with 0.1 M Na₂HPO₄, the precipitate formed taken up in water and dialyzed.